Complementary Longitudinal Serum Biomarkers to CA125 for Early Detection of Ovarian Cancer.

Early detection of ovarian cancer has the potential to impact mortality. A multimodal screening strategy where rising CA125 values over time, analyzed with the risk of ovarian cancer algorithm (ROCA), triggers transvaginal sonography and possible surgery has high sensitivity and specificity, but still fails to detect the 20% of early-stage cases that do not express CA125. Use of multiple biomarkers could detect cases missed by CA125. We have studied the sensitivity and lead time of a multi-marker panel (CA125, HE4, MMP-7, and CA 72-4) compared with CA125 alone. We used PRoBE design principles to select preclinical longitudinal specimens from 75 women (50 screen-positive, 25 screen-negative) who developed invasive epithelial ovarian cancer (3-5 serial specimens each) and 547 corresponding healthy controls (1-10 serial specimens each) from the ovarian cancer screening trial, UKCTOCS, in a blinded fashion. We measured the multi-marker concentrations in ultra-low serum volumes (16 µL) utilizing multiplexed bead-based immunoassays with low detection limits, high inter- and intra-assay precision, negligible cross-reactivity, and good correlation with standard immunoassays. While, at least one of the complementary biomarkers rose with CA125 in 44% (22/50) of screen-positive cases, there was no advantage in lead time over CA125. Therefore, we developed single-marker longitudinal algorithms (ROCA-like) to determine the presence of a change point to distinguish between the cases and controls. Using these algorithms, at 98% specificity, HE4 and CA72-4 identified 16% (4/25) of screen-negative cases, while MMP-7 identified none. Taken together, HE4 and CA72-4 show promise as complementary biomarkers to CA125 for longitudinal screening.

Validation of a Biomarker Panel and Longitudinal Biomarker Performance for Early Detection of Ovarian Cancer.

OBJECTIVES: Longitudinal multimarker combinations have the potential to improve sensitivity while maintaining the high specificity required for the early detection of ovarian cancer. The use of multiple markers to improve sensitivity over cancer antigen 125 (CA125) in longitudinal algorithms for early ovarian cancer detection requires the selection of markers with optimal discriminatory power and low longitudinal variance relative to disease-initiated changes. Our objective was to identify a multimarker panel suitable for ovarian cancer, where each individual marker has its own baseline, permitting longitudinal algorithm development. MATERIALS AND METHODS: In this retrospective study, we measured CA125, human epididymis protein 4 (HE4), matrix metalloproteinase-7 (MMP-7), CA72-4, CA19-9, CA15-3, carcinoembryonic antigen, and soluble vascular cell adhesion molecule (sVCAM) concentrations using immunoassays in pretreatment sera from 142 stage I ovarian cancer cases and 5 annual samples each from 217 healthy controls. After random division into training and validation sets, all possible biomarker combinations were explored exhaustively using linear classifiers to identify the panel with the greatest sensitivity for stage I disease at a high specificity of 98%. To evaluate longitudinal performance of the individual markers, the within-person over time and the between-person coefficient of variation (CV) were estimated. Hierarchical modeling across women of log-concentrations enabled the borrowing of information across subjects to moderate variance estimates given the small number of observations per subject. RESULTS: The 4-marker panel comprising CA125, HE4, MMP-7, and CA72-4 performed with the highest sensitivity (83.2%) at 98% specificity. The within-person CVs were lower for CA125, HE4, MMP-7, and CA72-4 (15%, 25%, 25%, and 21%, respectively) compared with their corresponding between-person CV (49%, 20%, 35%, and 84%, respectively) indicating baselines in healthy volunteers. After simple log-transformations, the within-volunteer variation across volunteers was modeled with a normal distribution permitting parsimonious hierarchical modeling. CONCLUSIONS: The multiplex panel chosen is suitable for the early detection of ovarian cancer and the individual markers have their own baseline permitting longitudinal algorithm development.

A multiplexable, microfluidic platform for the rapid quantitation of a biomarker panel for early ovarian cancer detection at the point-of-care.

Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large-scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p-BNC)-a multiplexable, microfluidic, modular platform-to quantify a novel multi-marker panel comprising CA125, HE4, MMP-7, and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4% and 10.5%, well below the biologic variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R(2) = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex platform. In a 31 patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with prediagnostic plasma from 2 cases and 4 controls. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening that takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels.

The emerging role of HE4 in the evaluation of epithelial ovarian and endometrial carcinomas.

HE4 (human epididymis protein 4) is overexpressed in both ovarian and endometrial cancers. Levels of the shed HE4 protein are elevated in sera from ovarian and endometrial cancer patients. HE4 is less frequently elevated than cancer antigen 125 (CA 125) in benign gynecologic conditions and is found in a fraction of endometrial and ovarian cancers that lack CA 125 expression. Consequently, HE4 has emerged as an important biomarker that complements CA 125 in discriminating between benign and malignant pelvic masses, monitoring response to treatment, and detecting recurrences of both ovarian and endometrial cancer. The "risk of ovarian malignancy algorithm" (ROMA) incorporates CA 125, HE4, and menopausal status to distinguish benign from malignant adnexal masses, and has been approved by the US Food and Drug Administration to aid in referring patients who are likely to have ovarian cancer to specially trained gynecologic oncologists for surgery. HE4 also promises to augment the sensitivity of CA 125 for detecting early-stage ovarian cancer. In this review, we discuss the discovery and biologic significance of HE4 and evaluate available evidence regarding the utility of HE4 as a biomarker for ovarian and endometrial cancer.